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Identification of Nevirapine-resistant HIV-1 in the Latent Reservoir following Single-dose Nevirapine
Megan Wind-Rotolo*1, C Durand1, L Cranmer1, A Reid1, J Siliciano1, M Doherty1, N Martinson1,2, S Eshleman1, T Quinn1, and R Siliciano1
1Johns Hopkins Univ, Baltimore, MD, US and 2Univ of Witwatersrand, Johannesburg, South Africa
Background: A single dose of nevirapine (NVP) given
to pregnant women during labor to prevent transmission of HIV-1 often leads to
the emergence of NVP-resistant virus in plasma. NVP-resistant virus has been
shown to fade from detection among plasma virus; however, the presence of this
virus in the latent reservoir in resting CD4+ T cells has not been
determined.
Methods: Plasma and peripheral blood mononuclear
cells (PBMC) were collected from women at least 6 months after they received single-dose
NVP. Highly purified resting CD4+ T cells were isolated and
activated in the presence of reverse transcriptase and integrase inhibitors to
prevent the completion of reverse transcription and integration in cells with
unintegrated virus. Virus from the latent reservoir was isolated from 51 women
(25 from Soweto, South Africa and 26 from Rakai, Uganda). Virus was also
isolated from 2 women who did not receive NVP. In addition, analysis of
replication competent virus released from the latent reservoir was performed on
a subset of these women. A highly sensitive mutation-specific assay (LigAmp)
was used to identify virus containing any of 3 NVP-resistance mutations (K103N,
Y181C, G190A) among virus from the latent reservoir and virus present in a
concurrent plasma sample.
Results: NVP-resistant virus was identified in the latent
reservoir of 12 of 51 women (23.5%) who received single-dose NVP. Of these, 7
women (13.7%) did not have NVP-resistant virus in the concurrent plasma sample.
One woman had 2 NVP-resistance mutations; one was present among both plasma
virus and virus from the latent reservoir, while the other mutation was present
only in virus from the latent reservoir. Virus containing the G190A mutation
was found most often among virus from the latent reservoir, followed by the
Y181C and K103N mutations. For selected samples, NVP-resistant virus isolated
from the latent reservoir was shown to be replication competent. Control samples
from women who did not receive NVP did not contain NVP-resistant virus.
Conclusions: These results provide the first
demonstration that NVP-resistant virus that arises following a single dose of
NVP can persist in the resting CD4+ T cell latent reservoir. The identification
of replication-competent NVP-resistant virus suggests that this virus could
re-emerge if a NNRTI is included in future HAART, providing a source of NVP-resistant
virus that could lead to future treatment failure and the development of
further drug-resistance mutations.
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